Macromolecule delivery to nanowells

ABSTRACT

Provided herein is technology relating to depositing and/or placing a macromolecule at a desired site for an assay and particularly, but not exclusively, to methods and systems for transporting a macromolecule such as a protein, a nucleic acid, or a protein:nucleic acid complex to an assay site, such as the bottom of a nanopore, a nanowell, or a zero mode waveguide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application is a divisional of U.S. application Ser. No.15/797,982 filed Oct. 30, 2017, which claims priority to U.S.application Ser. No. 14/369,642 filed Jun. 27, 2014, now U.S. Pat. No.9,803,231, issued Oct. 31, 2017, which is a national phase applicationunder 35 U.S.C. § 371 of PCT International Application No.PCT/US2012/072075, filed on Dec. 28, 2012, which claims priority to U.S.Provisional Application Ser. No. 61/581,508 filed Dec. 29, 2011, theentirety of which is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HDTRA1-10-C-0080awarded by Defense Threat Reduction Agency. The government has certainrights in the invention.

FIELD OF INVENTION

Provided herein is technology relating to depositing and/or placing amacromolecule at a desired site for an assay and particularly, but notexclusively, to methods and systems for transporting a macromoleculesuch as a protein, a nucleic acid, or a protein:nucleic acid complex toan assay site, such as the bottom of a nanopore, a nanowell, or a zeromode waveguide.

BACKGROUND

The massive parallelization of biological assays and realization ofsingle-molecule resolution have yielded profound advances in the waysthat biological systems are characterized and monitored and the way inwhich biological disorders are treated. Assays are able to interrogatethousands of individual molecules simultaneously, often in real time. Inparticular, the combination of solid state electronics technologies tobiological research applications has provided a number of importantadvances including, e.g., molecular array technology, i.e., DNA arrays(see, e.g., U.S. Pat. No. 6,261,776), microfluidic chip technologies(see e.g., U.S. Pat. No. 5,976,336), chemically sensitive field effecttransistors (ChemFETs), and other valuable sensor technologies.

These biochemical and medical assays often rely on the accurate andprecise positioning of individual assay components on a molecular scale.Thousands of nanoscale assays are often patterned on a substrate formacro-manipulation, analysis, and data recording. Accordingly, new toolsare needed to arrange and construct assay components with accuracy andprecision at a molecular resolution.

Components of Molecular Motors

One of the fundamental processes occurring in biological cells is activetransport of individual molecules (e.g., a macromolecule such as aprotein or a DNA) on a sub-micrometer scale. The simplest eukaryoticcell contains thousands of components that must be processed, packaged,sorted, and delivered to specific sites at specific times within thecell. These essential transport processes are carried out by motorproteins (e.g., kinesins and dyneins) that travel along microtubulesreaching into every corner of the cell. Motor proteins can beconceptualized as biological machines that transduce chemical energyinto mechanical forces and motion.

Microtubules, cytoskeletal fibers with a diameter of about 24 nm, havemultiple roles in the cell. Bundles of microtubules form cilia andflagella, which are whip-like extensions of the cell membrane that arenecessary for sweeping materials across an epithelium and for swimmingof sperm, respectively. Marginal bands of microtubules in red bloodcells and platelets are important for these cells' pliability.Organelles, membrane vesicles, and proteins are transported in the cellalong tracks of microtubules. For example, microtubules run throughnerve cell axons, allowing bi-directional transport of materials andmembrane vesicles between the cell body and the nerve terminal. Failureto supply the nerve terminal with these vesicles blocks the transmissionof neural signals. Microtubules are also critical to chromosomalmovement during cell division. Both stable and short-lived populationsof microtubules exist in the cell.

Microtubules are polymers of GTP-binding tubulin protein subunits. Eachsubunit is a heterodimer of alpha- and beta-tubulin, multiple isoformsof which exist. The hydrolysis of GTP is linked to the addition oftubulin subunits at the end of a microtubule. The subunits interacthead-to-tail to form protofilaments; the protofilaments interactside-to-side to form a microtubule. A microtubule is polarized, one endringed with alpha-tubulin (e.g., the “− end”) and the other withbeta-tubulin (e.g., the “+end”), and the two ends differ in their ratesof assembly. Generally, each microtubule is composed of 13protofilaments although 11 or 15 protofilament-microtubules aresometimes found. Cilia and flagella contain doublet microtubules.

Methods have been developed for manipulation of microtubules.Microtubules can be routinely reassembled in vitro from tubulin purifiedfrom bovine brains. For example, the nucleation, assembly, anddisassembly reactions of microtubules have been well characterized in,e.g., L. U. Cassimeris et al., Dynamic Instability of Microtubules, 7Bioessays 149 (1988). More recently, considerable progress has been madetoward producing recombinant tubulin in yeast. See A. Davis et al.,Purification and Biochemical Characterization of Tubulin from theBudding Yeast Saccharomyces cerevisiae, 32 Biochemistry 8823 (1993).

The motor protein, kinesin, was discovered in 1985 in squid axoplasm. R.D. Vale et al., Identification of a Novel Force-generating Protein,Kinesin, Involved in Microtubule based Motility, 42 Cell 39-50 (1985).It has been discovered that kinesin is just one member of a very largefamily of motor proteins. E.g., S. A. Endow, The Emerging Kinesin Familyof Microtubule Motor Proteins, 16 Trends Biochem. Sci. 221 (1991); L. S.B. Goldstein, The Kinesin Superfamily: Tails of Functional Redundancy, 1Trends Cell Biol. 93 (1991); R. J. Stewart et al., Identification andPartial Characterization of Six Members of the Kinesin Superfamily inDrosophila. 88 Proc. Nat'l Acad. Sci. USA 8470 (1991). Other motorproteins include dynein, e.g., M.-G. Li et al., Drosophila CytoplasmicDynein, a Microtubule Motor that is Asymmetrically Localized in theOocyte, 126 J. Cell Biol. 1475-93 (1994), and myosin, e.g., T. Q. P.Uyeda et al, 214 J. Molec. Biol. 699-710 (1990). Kinesin, dynein, andrelated proteins move along microtubules, whereas myosin moves alongactin filaments.

Kinesins are motor proteins that act on microtubules and that typicallymove toward the +end of the microtubule. The prototypical kinesinmolecule is involved in the transport of membrane-bound vesicles andorganelles. This function is particularly important for axonal transportin neurons. Kinesin is also important in all cell types for thetransport of vesicles from the Golgi complex to the endoplasmicreticulum. This role is critical for maintaining the identity andfunctionality of these secretory organelles.

Kinesins define a ubiquitous, conserved family of over 50 proteins thatcan be classified into at least 8 subfamilies based on primary aminoacid sequence, domain structure, velocity of movement, and cellularfunction. (Reviewed in Moore, J. D. and S. A. Endow (1996) Bioessays18:207-219; and Hoyt, A. M. (1994) Curr. Opin. Cell Biol. 6:63-68.) Theprototypical kinesin molecule is a heterotetramer composed of two heavypolypeptide chains (KHCs) and two light polypeptide chains (KLCs). TheKHC subunits are typically referred to as “kinesin.” KHC is about 1000amino acids in length (having a mass of about 120 kDa) and KLC is about550 amino acids in length (having a mass of about 60 kDa). Two KHCsdimerize to form a rod-shaped molecule with three distinct regions ofsecondary structure. At one end of the molecule is a globular motordomain that functions in ATP hydrolysis and microtubule binding. Kinesinmotor domains are highly conserved and share over 70% identity. Beyondthe motor domain is an alpha-helical coiled-coil region that mediatesdimerization. At the other end of the molecule is a fan-shaped tail thatassociates with molecular cargo. The tail is formed by the interactionof the KHC C-termini with the two KLCs.

The kinesin heavy chains comprise three structural domains: (a) anamino-terminal head domain, which contains the sites for ATP andmicrotubule binding and for motor activity; (b) a middle or stalkdomain, which may form an alpha-helical coiled coil that entwines twoheavy chains to form a dimer; and (c) a carboxyl-terminal domain, whichprobably forms a globular tail that interacts with the light chains andpossibly with vesicles and organelles. Kinesin and kinesin-like proteinsare all related by sequence similarity within an approximately 340-aminoacid region of the head domain, but outside of this conserved regionthey show no sequence similarity.

The motility activity of purified kinesin on microtubules has beendemonstrated in vitro. R. D. Vale et al., Identification of a NovelForce-generating Protein, Kinesin, Involved in Microtubule-basedMotility, 42 Cell 39-50 (1985). Further, full-length kinesin heavy chainand several types of truncated kinesin heavy chain molecules produced inE. coli are also capable of generating in vitro microtubule motility. J.T. Yang et al., Evidence that the Head of Kinesin is Sufficient forForce Generation and Motility. In Vitro, 249 Science 42 (1990); R. J.Stewart et al, Direction of Microtubule Movement is an Intrisic Propertyof the Motor Domains of Kinesin Heavy Chain and Drosophila NCD Protein,90 Proc. Nat'l Acad. Sci. USA 5209-13 (1993). The kinesin motor domainhas also been shown to retain motor activity in vitro after geneticfusion to several other proteins including spectrin, J. T. Yang et al.,The Head of Kinesin is Sufficient for Force Generation and Motility InVitro, 249 Science 42 (1990), glutathione S-transferase, R. J. Stewartet al., Direction of Microtubule Movement is an Intrinsic Property ofthe NCD and Kinesin Heavy Chain Motor Domains, 90 Proc. Nat'l Acad. Sci.USA 5209 (1993), and biotin carboxyl carrier protein, E. Berliner,Microtubule Movement by a Biotinated Kinesin Bound to aStreptavidincoated Surface, 269 J. Biol. Chem. 8610 (1994).

In addition to kinesins, dyneins are also motor proteins that bind toand act on microtubules and typically move toward the − end of themicrotubule. Two classes of dyneins, cytosolic and axonemal, have beenidentified. Cytosolic dyneins are responsible for translocation ofmaterials along cytoplasmic microtubules, for example, for transportfrom the nerve terminal to the cell body and transport of endocyticvesicles to lysosomes. As well, viruses often take advantage ofcytoplasmic dyneins to be transported to the nucleus and establish asuccessful infection. Sodeik, B. et al. 136 J. Cell Biol. 1007-21(1997). Virion proteins of herpes simplex virus 1, for example, interactwith the cytoplasmic dynein intermediate chain. Ye, G. J. et al. 74 J.Virol. 1355-63 (2000). Cytoplasmic dyneins are also reported to play arole in mitosis. Axonemal dyneins are responsible for the beating offlagella and cilia. Dynein on one microtubule doublet walks along theadjacent microtubule doublet. This sliding force produces bending thatcauses the flagellum or cilium to beat. Dyneins have a native massbetween 1000 and 2000 kDa and contain either two or threeforce-producing heads driven by the hydrolysis of ATP. The heads arelinked via stalks to a basal domain which is composed of a highlyvariable number of accessory intermediate and light chains. Cytoplasmicdynein is the largest and most complex of the motor proteins.

Myosins are actin-activated ATPases, found in eukaryotic cells, thatcouple hydrolysis of ATP with motion. Myosin provides the motor functionfor muscle contraction and intracellular movements such as phagocytosisand rearrangement of cell contents during mitotic cell division(cytokinesis). The contractile unit of skeletal muscle, termed thesarcomere, consists of highly ordered arrays of thin actin-containingfilaments and thick myosin-containing filaments. Crossbridges formbetween the thick and thin filaments, and the ATP-dependent movement ofmyosin heads within the thick filaments pulls the thin filaments,shortening the sarcomere and thus the muscle fiber.

Myosins are composed of one or two heavy chains and associated lightchains. Myosin heavy chains contain an amino-terminal motor or headdomain, a neck that is the site of light-chain binding, and acarboxy-terminal tail domain. The tail domains may associate to form analpha-helical coiled coil. Conventional myosins, such as those found inmuscle tissue, are composed of two myosin heavy-chain subunits, eachassociated with two light-chain subunits that bind at the neck regionand play a regulatory role. Unconventional myosins, believed to functionin intracellular motion, may contain either one or two heavy chains andassociated light chains. There is evidence for about 25 myosin heavychain genes in vertebrates, more than half of them unconventional.

Actin is the most abundant intracellular protein in the eukaryotic cell.Actin filaments interact with myosin in muscles and provide a frameworkto support the plasma membrane and determine cell shape. In musclecells, thin filaments containing actin slide past thick filamentscontaining the motor protein myosin during contraction. Microfilamentsare the polymerized form of actin and are vital to cell locomotion, cellshape, cell adhesion, cell division, and muscle contraction. Assemblyand disassembly of the microfilaments allow cells to change theirmorphology. Human cells contain six isoforms of actin. The threealpha-actins are found in different kinds of muscle, nonmusclebeta-actin, and nonmuscle gamma-actin are found in nonmuscle cells, andanother gamma-actin is found in intestinal smooth muscle cells. G-actin,the monomeric form of actin, polymerizes into polarized, helical F-actinfilaments, accompanied by the hydrolysis of ATP to ADP. A family ofactin-related proteins exist that are not part of the actincytoskeleton, but rather associate with microtubules and dynein.

Zero Mode Waveguides

In some assays, molecules are confined in a series, array, or otherarrangement of small holes, pores, or wells, for example, a zero modewaveguide (ZMW). ZMW arrays have been applied to a range of biochemicalanalyses and have found particular usefulness for genetic analysis. ZMWstypically comprise a nanoscale core, well, or opening disposed in anopaque cladding layer that is disposed upon a transparent substrate,e.g., a circular hole in an aluminum cladding film deposited on a clearsilica substrate. J. Korlach et al., Selective aluminum passivation fortargeted immobilization of single DNA polymerase molecules in zero-modewaveguide nanostructures. 105 PNAS 1176-81 (2008). A typical ZMW hole is˜70 nm in diameter and ˜100 nm in depth. ZMW technology allows thesensitive analysis of single molecules because, as light travels througha small aperture, the optical field decays exponentially inside thechamber. That is, due to the narrow dimensions of the well,electromagnetic radiation that is of a frequency above a particularcut-off frequency will be prevented from propagating all the way throughthe core. Notwithstanding the foregoing, the radiation will penetrate alimited distance into the core, providing a very small illuminatedvolume within the core. By illuminating a very small volume, one canpotentially interrogate very small quantities of reagents, including,e.g., single molecule reactions. The observation volume within anilluminated ZMW is ˜20 zeptoliters (20×10⁻²¹ liters). Within thisvolume, the activity of DNA polymerase incorporating a single nucleotidecan be readily detected.

By monitoring reactions at the single molecule level, one can preciselyidentify and/or monitor a given reaction. The technology is not limitedin the types of single molecule interactions that can be observed (e.g.,a non-limiting list is protein-protein, protein-DNA, DNA-DNA, DNA-RNA,RNA-RNA, protein-RNA, lipid-lipid, protein-lipid, enzyme-substrate,enzyme-intermediate, enzyme-product, enzyme-metabolite, enzyme-cofactor,enzyme-inhibitor, etc.). In particular, the technology is the basis fora particularly promising field of single molecule DNA sequencingtechnology that monitors the molecule-by-molecule (e.g.,nucleotide-by-nucleotide) synthesis of a DNA strand in atemplate-dependent fashion by a single polymerase enzyme (e.g., SingleMolecule Real Time (SMRT) DNA Sequencing as performed, e.g., by aPacific Biosciences RS Sequencer (Pacific Biosciences, Menlo Park,Calif.)). See, e.g., U.S. Pat. Nos. 7,476,503; 7,486,865; 7,907,800; and7,170,050; and U.S. patent application Ser. Nos. 12/553,478, 12/767,673;12/814,075; 12/413,258; and Ser. No. 12/413,466, each incorporatedherein by reference in its entirety for all purposes. See also, Eid, J.et al. 2009. “Real-time DNA sequencing from single polymerasemolecules”, 323 Science: 133-38 (2009); Korlach, J. et al. “Long,processive enzymatic DNA synthesis using 100% dye-labeled terminalphosphate-linked nucleotides”, 27 Nucleosides, Nucleotides & NucleicAcids: 1072-82 (2008); Lundquist, P. M. et al., “Parallel confocaldetection of single molecules in real time”, 33 Optics Letters: 1026-28(2008); Korlach, J. et al., “Selective aluminum passivation for targetedimmobilization of single dna polymerase molecules in zero-mode waveguidenanostructures”, 105 Proc Natl Acad Sci USA: 1176-81 (2008); Foquet, M.et al., “Improved fabrication of zero-mode waveguides forsingle-molecule detection”, 103 Journal of Applied Physics (2008); andLevene, M. J. et al. “Zero-mode waveguides for single-molecule analysisat high concentrations”, 299 Science: 682-86 (2003), each incorporatedherein by reference in its entirety for all purposes.

In conventional use, placing components in the wells of the ZMW relieson simple diffusion to deliver components (e.g., macromolecules such asDNA polymerase and/or DNA and/or DNA/DNA polymerase complexes) to thedesired site (e.g., the bottom of the ZMW well) in the zero modewaveguides. As a result, a significant amount of the macromolecule(e.g., the DNA polymerase/DNA complex) needs to be added to the ZMWs toachieve a critical mass sufficient enough to drive the diffusion of thecomplexes into the bottom of the wells. This process is not efficient:e.g., only a fraction of the complexes reaches the desired sites in thewells and incubation times are required to position the assay componentsin the proper sites. Moreover, extensive incubation times (e.g., 4 ormore hours) are required to form the complexes to be delivered to theZMWs.

SUMMARY

Provided herein is technology for the active transport of assaycomponents (e.g., a macromolecule such as a DNA, DNA polymerase, DNA/DNApolymerase complex, a protein, etc.) to a desired site for an assay(e.g., the bottom of a ZMW well). The technology provides compositions,methods, and systems using actin filaments or microtubules that arebound to the bottom of a zero mode waveguide. The actin filaments ormicrotubules serve as transport guides for the macromolecules (e.g., theDNA polymerase or DNA polymerase/DNA complex).

These exemplary embodiments are not intended to limit the technology.Indeed, it is intended to be understood that the technology is widelyapplicable to any instance in which a molecular cargo needs to betransported to a site. Additional embodiments will be apparent topersons skilled in the relevant art based on the teachings containedherein.

Accordingly, provided herein is technology providing methods,compositions, and systems for the delivery of macromolecules to adesired site. In particular, some aspects of the technology provide fora composition for guiding a macromolecule to a site, wherein thecomposition comprises a transport guide comprising a loading end and adelivery end at the site; a molecular motor that binds to and movesalong the transport guide; and a macromolecule comprising a linkingdomain, wherein the linking domain links the macromolecule to themolecular motor. Some embodiments further provide that an assay isperformed at the site, e.g., some embodiments provide that DNAsequencing occurs at the site. In aspects of the technology related toDNA sequencing, the compositions further comprise a phospholinkednucleotide.

In some embodiments, the composition finds use in delivering amacromolecule to a well, pore, or nanoscale assay site such as a zeromode waveguide. Accordingly, in some embodiments, the site is in a zeromode waveguide and in some embodiments the site is in a nanowell.

Some embodiments of the technology provide that the transport guide is amicrotubule and the molecular motor is one or more of a kinesin and adynein. Various types of kinesins are appropriate for the technology andthus the technology is not limited by the particular embodimentsdescribed herein. For example, in some embodiments the kinesin is achromokinesin (e.g., a KIN N chromokinesin) and in some embodimentsother kinesins are provided in the described compositions. In someembodiments, the transport guide is an actin filament and the molecularmotor is a myosin.

The technology finds use in the transport of macromolecules, forinstance in some embodiments the macromolecule comprises an enzyme(e.g., a DNA polymerase, an RNA polymerase, a reverse transcriptase) andin some embodiments the macromolecule comprises a nucleic acid (e.g., aDNA or an RNA).

In some embodiments, an enzyme/nucleic acid complex is transported tothe site and in some embodiments the composition further comprises anenzyme (e.g., a polymerase, e.g., a DNA or an RNA polymerase) at thesite. Some embodiments provide compositions further comprising an anchorto maintain the macromolecule at the site.

The technology provides for the attachment, association, linking,binding, etc. of the molecular motor (e.g., the myosin, dynein, kinesin)to the macromolecule. Thus, in some embodiments a composition isprovided in which the linking domain mediates a covalent interactionwith the molecular motor and in some embodiments the linking domainmediates a non-covalent interaction with the molecular motor. Forexample, some embodiments provide that the linking domain comprises amyosin binding domain and/or a microtubule associated protein bindingdomain. Particular embodiments provide that the macromolecule is a DNAcomprising a sequence specifically bound by the chromokinesin. Someembodiments comprise other linking domains and/or systems, for example,some embodiments provide a composition further comprising astreptavidin, a biotinylated oligonucleotide, and a linking domain thatis a streptavidin binding domain. Such embodiments additionally providethat the biotinylated oligonucleotide is complementary to a libraryadaptor sequence.

In some embodiments, the transport guide is stabilized, for example, ina composition comprising a phalloidin, and/or a paclitaxel (e.g., ataxol). In some embodiments, the transport guide is disassembled and/ordestabilized by a cytochalasin.

Provided herein are compositions related to delivering macromolecules toa site, e.g., to perform a biological assay such as DNA or RNAsequencing. For example, the technology provides embodiments ofcompositions for delivering a nucleic acid (e.g., a DNA or an RNA), anenzyme (e.g., a DNA polymerase, an RNA polymerase, or a reversetranscriptase), or an enzyme/nucleic acid complex to the bottom of azero mode waveguide, wherein the composition comprises an actin filamentor a microtubule with one end attached to a bottom of a zero modewaveguide well; a myosin, kinesin, or dynein for traveling along theactin filament or microtubule; and a nucleic acid, an enzyme (such as aDNA polymerase, RNA polymerase, or a reverse transcriptase), or anenzyme/DNA complex attached to the myosin, kinesin, or dynein, whereinthe myosin, kinesin, or dynein transports the nucleic acid, enzyme, orenzyme/nucleic acid complex to the bottom of the zero mode waveguidewell for single-molecule real-time sequencing.

The technology provides for embodiments of methods, e.g., for deliveringa macromolecule to a site, wherein the method comprises maintaining anend of a transport guide at the site; providing a molecular motoradapted for binding the transport guide and moving along the transportguide; and linking the macromolecule to the molecular motor. In someembodiments, the macromolecule is delivered to the site for an assay(e.g., an assay is performed at the site). Some embodiments provide thatDNA or RNA sequencing occurs at the site, which, in some embodimentsfurther comprise providing a phospholinked nucleotide. Assays andnucleic acid sequencing are performed in some embodiments in a nanopore,nanowell, and, in some embodiments, in a zero-mode waveguide.

In some embodiments, the transport guide is a microtubule and themolecular motor comprises a kinesin and/or a dynein. Particularembodiments provide that the kinesin is a chromokinesin (e.g., a KIN Nchromokinesin). An aspect of the technology provides embodiments inwhich the transport guide is an actin filament and the molecular motoris a myosin.

The methods provided find use in various embodiments of the technologythat transport a macromolecule. For example, in some embodiments, themacromolecule comprises an enzyme such as DNA polymerase or reversetranscriptase and in some embodiments the macromolecule comprises anucleic acid such as DNA or RNA. Some embodiments provide the enzyme atthe site.

The transport guide provides for the transport of a macromolecule to aparticular site, e.g., for an assay or nucleic acid sequencing. In someembodiments, the methods comprise maintaining the end of the transportguide at the site, e.g., in a method that comprises attaching the end ofthe transport guide to the site.

Some embodiments provide that the linking comprises providing a covalentinteraction of the macromolecule and the molecular motor and someembodiments provide that the linking comprises providing a non-covalentinteraction of the macromolecule and the molecular motor. In particularembodiments, the linking comprises providing a domain comprising amyosin binding domain and/or a microtubule associated protein bindingdomain. For example, in some embodiments, the macromolecule is a DNAcomprising a sequence specifically bound by a chromokinesin. In someembodiments, the methods comprise providing a streptavidin, abiotinylated oligonucleotide, and a streptavidin binding domain. Inadditional embodiments, the biotinylated oligonucleotide iscomplementary to a library adaptor sequence in the target nucleic acid.Some embodiments of the methods provide that the transport guide isstabilized by providing a member comprising a phalloidin or apaclitaxel. In some embodiments, the transport guide is disassembledand/or destabilized by a cytochalasin.

Provided herein are methods related to delivering macromolecules to asite, e.g., to perform a biological assay and/or nucleic acidsequencing. For example, the technology provides embodiments of methodsfor delivering a nucleic acid (e.g., a DNA or an RNA), an enzyme (e.g.,a DNA polymerase, an RNA polymerase, or a reverse transcriptase), or anenzyme/nucleic acid complex to the bottom of a zero mode waveguide,wherein the method comprises attaching an actin filament or amicrotubule to a bottom of a zero mode waveguide well; providing amyosin, kinesin, or dynein for binding and traveling along the actinfilament or microtubule; linking an enzyme (e.g., a DNA polymerase, anRNA polymerase, or a reverse transcriptase), a nucleic acid (e.g., a DNAor an RNA), or an enzyme/nucleic acid complex to the myosin, kinesin, ordynein; and transporting the enzyme (e.g., a DNA polymerase, an RNApolymerase, or a reverse transcriptase), the nucleic acid (e.g., a DNAor an RNA), or the enzyme/nucleic acid complex to the bottom of the zeromode waveguide well for single-molecule real-time sequencing.

The methods and compositions provided herein find use in systems fortransporting a macromolecule to a site, e.g., embodiments of a systemcomprising a transport guide (e.g., an actin filament or a microtubule);a molecular motor for transporting the macromolecule along the transportguide; and a linking domain for linking the macromolecule to themolecular motor.

Some embodiments provide a system for sequencing a nucleic acid molecule(e.g., a DNA, RNA, etc.), the system comprising a transport guidecomprising an actin filament or a microtubule; a molecular motor fortransporting the macromolecule along the transport guide; a linkingdomain for linking the macromolecule to the molecular motor; and aphospholinked nucleotide. Some embodiments provide that the sequencingoccur in a zero mode waveguide and thus the systems according to someembodiments provide a zero mode waveguide. Some embodiments of thesystems further comprise an anchor to maintain the macromolecule at thesite. The systems comprise a molecular motor, e.g., in some embodimentsthe systems comprise a kinesin, a dynein, or a myosin.

The technologies provided herein find use in methods for the manufactureof an assay component, for example, a method of manufacturing an assaycomponent, the method comprising attaching one end of a transport guideto a site; linking a macromolecule to a molecular motor; binding themolecular motor to the transport guide; and transporting themacromolecule to the site. The methods find use in manufacturing acomponent, surface, device, etc. for performing an assay, e.g., abiological assay to measure the interaction of molecules or.Accordingly, in some embodiments, an assay is performed at the site.Moreover, the methods find use in manufacturing a component, surface,device, etc. for performing nucleic acid sequencing. Accordingly, insome embodiments, nucleic acid sequencing occurs at the site. Thetechnologies find use in the manufacture of components comprisingstructures for performing assays on a molecular scale, for instance ananowell, a nanopore, or a zero mode waveguide. Thus, in someembodiments, the site is in a zero-mode waveguide and in someembodiments the site is in a nanowell.

It is contemplated that the technologies described herein make use of anappropriate transport guide and molecular motor and it is not to beconstrued that the technologies are limited to any particular transportguide and/or molecular motor disclosed herein. Embodiments of thetechnology comprise exemplary methods wherein the transport guide is amicrotubule and the molecular motor comprises a kinesin and/or a dynein.In a particular embodiment, the kinesin is a chromokinesin. In otherexemplary embodiments, the transport guide is an actin filament and themolecular motor is a myosin. It is to be understood that thetechnologies are applicable to transport, place, localize, or otherwiseposition any macromolecule at a site to manufacture an assay component.In some embodiments, the macromolecule comprises an enzyme such as DNApolymerase or reverse transcriptase and in some embodiments themacromolecule comprises a nucleic acid such as a DNA or an RNA. In someembodiments, the methods further comprise placing the enzyme at thesite. In some embodiments, the macromolecule is transported to the siteand anchored at the site; as such, in some embodiments, the methodsprovided further comprise anchoring the macromolecule at the site.

One of skill in the art understands that many technologies are availableto link molecules and macromolecules. In some embodiments, the linkingcomprises covalently interacting with the molecular motor and in someembodiments the linking comprises non-covalently interacting with themolecular motor. In particular embodiments, the linking is mediated by amember comprising a myosin binding domain and/or a microtubuleassociated protein binding domain. In other embodiments, themacromolecule comprises a sequence specifically bound by thechromokinesin. In some embodiments, the linking is mediated by astreptavidin, a biotinylated oligonucleotide, and a streptavidin bindingdomain. In a specific embodiment, the biotinylated oligonucleotide iscomplementary to a library adaptor sequence. Embodiments of thetechnology comprise stabilizing the transport guide with a compositioncomprising a phalloidin and/or a paclitaxel. In some embodiments, thetransport guide is disassembled and/or destabilized by a cytochalasin.

The technology provided herein finds use in methods for delivering anucleic acid molecule (e.g., a DNA or an RNA), an enzyme (e.g., a DNApolymerase, RNA polymerase, or reverse transcriptase), or anenzyme/nucleic acid complex to the bottom of a zero mode waveguide,wherein the method comprises attaching an end of an actin filament or amicrotubule to a bottom of a zero mode waveguide well; linking thenucleic acid molecule (e.g., a DNA or an RNA), the enzyme (e.g., a DNApolymerase, RNA polymerase, or reverse transcriptase), or theenzyme/nucleic acid complex to a myosin, a kinesin, or a dynein; bindingthe myosin, the kinesin, or the dynein to the actin filament or themicrotubule; and transporting the nucleic acid molecule (e.g., a DNA oran RNA), the enzyme (e.g., a DNA polymerase, RNA polymerase, or reversetranscriptase), or the enzyme/nucleic acid complex to the bottom of thezero mode waveguide, wherein the myosin, the kinesin, or the dyneintransports the nucleic acid molecule (e.g., a DNA or an RNA), the enzyme(e.g., a DNA polymerase, RNA polymerase, or reverse transcriptase), orthe enzyme/nucleic acid complex to the bottom of the zero mode waveguidewell for single-molecule real-time DNA (or RNA) sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is a drawing showing an embodiment of the technology comprising aDNA polymerase engineered to contain a kinesin binding domain.

FIG. 2 is a drawing showing an embodiment of the technology comprising aDNA adaptor engineered to contain a chromokinesin binding sequence.

FIG. 3 is a drawing showing an embodiment of the technology comprising akinesin covalently linked to an oligonucleotide that is complementary toa library adaptor sequence.

DETAILED DESCRIPTION

Provided herein is technology for the active transport of assaycomponents (e.g., a macromolecule such as a DNA, DNA polymerase, DNA/DNApolymerase complex, a protein, etc.) to a desired site for an assay(e.g., the bottom of a ZMW well). For example, the technology providescompositions, methods, and systems using actin filaments or microtubulesthat are bound to the bottom of a zero mode waveguide. The actinfilaments or microtubules serve as transport guides for themacromolecules (e.g., the DNA polymerase or DNA polymerase/DNA complex).

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein, the term “site” is used to refer to a location in threedimensional space on a molecular scale that is of interest for thetechnology provided herein (e.g., where a measurement occurs and/or theposition of a molecule). In some embodiments, the site is on a surfaceor on a substrate and in some embodiments the site is in a solution. Forexample, in some embodiments the site comprises a concentration orcollection of molecules (biological molecules or other chemicals) thatinteract, for example in a biochemical (e.g., enzymatic) reaction (e.g.,DNA synthesis). In some embodiments, an interaction of molecules occursat the site and the interaction is measured, quantified, assessed,and/or otherwise evaluated. In some embodiments, the reactants and/orproducts consumed and/or produced at the site are measured, quantified,assessed, and/or otherwise evaluated. In some embodiments, the site isthe position in space of a single molecule. In some embodiments, thesite is the position of a single atom. In some embodiments, the site isat the bottom of a nanowell or zero mode waveguide where amacromolecular interaction or biochemical reaction is monitored.

As used herein, the term “transport guide” is used to refer to amolecular structure that guides the transport of a molecule in threedimensional space, e.g., by a molecular motor. A transport guideprovides a substrate for movement of a transporter such as a molecularmotor. In some embodiments, a tubulin or actin filament is a transportguide. A transport guide may be thought of as a rail of a moleculartrain.

As used herein, the term “linking domain” is used to refer to a domainor moiety of a molecule or macromolecule that mediates an associationwith another interacting partner, e.g., a molecule, macromolecule, oratom. The linking domain may be a native domain of the molecule ormacromolecule or may be engineered into the molecule or macromolecule.The linking domain may have other functions in addition to mediating anassociation with another interacting partner (atom, molecule,macromolecule). In some embodiments, the linking domain interactsdirectly with another molecule or macromolecule; in some embodiments,the interacting molecules or macromolecules each comprise a linkingdomain and the association between the molecules or macromolecules ismediated by the interaction of linking domains present on each moleculeor macromolecule. In some embodiments, one or more additional moleculesor macromolecules may bridge the interaction between a linking domainand a molecule or macromolecule or between the linking domains of one ormore molecules or macromolecules. For example, in some embodiments oneinteracting partner comprises a linking domain that is a streptavidinand the other interacting partner comprises a linking domain that is abiotin. In some embodiments, one linking domain is a streptavidinbinding protein, another linking domain is a biotin moiety, and theinteraction between the two is mediated by a bridging streptavidin.Additional examples are, in some embodiments, linking domains comprisinga DNA-binding domain. For example, a chromokinesin contains both akinesin motor-like domain and a DNA-binding domain (e.g., abasic-leucine zipper). Accordingly, a chromokinesin (e.g., a KIN Nchromokinesin) binds a specific DNA sequence. For example, DrosophiliaNOD binds the AATAT repeats of the 1.672 satellite DNA (S. Bonaccorsiand A. Lohe. “Fine Mapping of Satellite DNA Sequences along the YChromosome of Drosophila melanogaster: Relationships between SatelliteSequences and Fertility Factors”. 1991 Genetics 129(1): 177-89). HumanKID binds to cerb2 promoter sequences (Tokai et al., “Kid, a novelkinesin-like DNA binding protein, is localized to chromosomes and themitotic spindle” 1996 EMBO J 15(3): 457-67). See also Afshar et al.,“DNA binding and meiotic chromosomal localization of the Drosophila nodkinesin-like protein” 1995 Cell 81(1): 129-38, all of which areincorporated herein by reference in their entireties.

Specific protein-protein interactions can be used to for linkingdomains, e.g., antibody-antigen or antibody-epitope, myosin bindingdomain-myosin, and other specific binding partners known in the art ofmolecular biology. In some embodiments, the interactions or associationsare mediated by a covalent link (e.g., a chemical bond) and in someembodiments the interactions or associations are mediated by anoncovalent link or binding.

As used herein, the term “molecular motor” refers to a molecule,macromolecule, or molecular assembly that utilizes chemical energy togenerate mechanical force.

As used herein, a “phospholinked nucleotide” is a nucleotide having alabel (e.g., a fluor or dye) attached to a phosphate (e.g., the terminalphosphate, e.g., the terminal phosphate of the NTP triphosphate chain).Upon incorporation of the labeled phospholinked nucleotide into thegrowing synthesized DNA molecule, the label (e.g., the flour or dye) iscleaved from the NTP.

As used herein, an “anchor” is a molecule or macromolecule thatreversibly or irreversibly attaches, immobilizes, localizes, orassociates a molecule, macromolecule, or atom to a surface or substrate.

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably. A“protein” or “polypeptide” encoded by a gene is not limited to the aminoacid sequence encoded by the gene, but includes post-translationalmodifications of the protein.

Where the term “amino acid sequence” is recited herein to refer to anamino acid sequence of a protein molecule, “amino acid sequence” andlike terms, such as “polypeptide” or “protein” are not meant to limitthe amino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule, but is intended to includeother forms such as “portions”, “fragments”, “variants”, and “mutants”as defined below. Furthermore, an “amino acid sequence” can be deducedfrom the nucleic acid sequence encoding the protein. The term “portion”when used in reference to a protein (as in “a portion of a givenprotein”) refers to fragments of that protein. The fragments may rangein size from four amino acid residues to the entire amino sequence minusone amino acid (for example, the range in size includes 4, 5, 6, 7, 8,9, 10, or 11 . . . amino acids up to the entire amino acid sequenceminus one amino acid).

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions(e.g., additions), or both. Guidance in determining which and how manyamino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

The term “domain” when used in reference to a polypeptide refers to asubsection of the polypeptide which possesses a unique structural and/orfunctional characteristic; typically, this characteristic is similaracross diverse polypeptides. The subsection typically comprisescontiguous amino acids, although it may also comprise amino acids whichact in concert or which are in close proximity due to folding or otherconfigurations. Examples of a protein domain include transmembranedomains and the glycosylation sites.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide. Thus, “a nucleotide comprising at least aportion of a gene” may comprise fragments of the gene or the entiregene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kbp on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

The terms “an oligonucleotide having a nucleotide sequence encoding agene” or “a nucleic acid sequence encoding” a specified polypeptiderefer to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNA,or RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “5′-A-G-T-3′” is complementary to the sequence“3′-T-C-A-5′.” Complementarity may be “partial,” in which only some ofthe nucleic acids' bases are matched according to the base pairingrules. Or, there may be “complete” or “total” complementarity betweenthe nucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

Embodiments of the Technology

In some embodiments, the technology comprises a polymerase (e.g., a DNApolymerase) or other enzyme engineered to contain either a myosinbinding domain, which binds the myosin protein, or a microtubuleassociated protein binding domain, which binds kinesin or dynein, or anyother binding domain associated with a transport guide molecule. In someembodiments, a polymerase/nucleic acid complex is formed from anengineered polymerase and a nucleic acid molecule, and then thepolymerase/nucleic acid complex is incubated with the appropriate motorprotein (e.g., myosin if using actin filaments; kinesin or dynein ifusing microtubules) for binding. The polymerase/nucleic acid/motorprotein complex is then added to the ZMW where it binds and travels downthe actin filament or microtubule to the bottom of the well. Motorproteins are known to travel in only one direction: For example, kinesinproteins travel to the positive (+) end of microtubules and dyneinstravel to the negative (−) end of microtubules. In embodiments whereinkinesin is used, the positive end of the microtubule is anchored in thebottom of the ZMW and kinesin carries the cargo (DNA polymerase/DNAcomplex) to the bottom of the well. In embodiments wherein dynein isused, the negative end of the microtubule is anchored in the bottom ofthe ZMW and dynein carries the cargo (DNA polymerase/DNA complex) to thebottom of the well.

In another aspect of the technology, ZMWs are pretreated with polymeraseunder conditions that maximize polymerase binding to the well (e.g.,ZMWs are incubated with polymerase under optimal conditions for bindingfor a time sufficient for binding). Microtubules or actin filaments areanchored to the bottom of the ZMWs. Chromokinesins, a specific type ofkinesin motor protein that binds specific DNA sequences (see, e.g.,Yajima J, E, et al. (2003). “The human chromokinesin Kid is a plusend-directed microtubule-based motor”. 22 EMBO J.: 1067-74 (2003);Tokai-Nishizumi N, et al. “The chromokinesin Kid is required formaintenance of proper metaphase spindle size” 16 Mol. Biol. Cell 5455-63(2006)), are incubated with DNA libraries that contain the chromokinesinbinding sequences (these sequences are incorporated into the libraryadaptor sequences). The chromokinesin/DNA complexes are then loaded ontothe ZMWs containing the microtubules and DNA polymerase. Thechromokinesin/DNA complex travels down the microtubule, delivering thecomplex to the DNA polymerase at the bottom of the well.

In related aspects of the technology, ZMWs are pretreated withpolymerase under conditions that maximize polymerase binding to the well(e.g., ZMWs are incubated with polymerase under optimal conditions forbinding for a time sufficient for binding). Microtubules or actinfilaments are anchored to the bottom of the ZMWs. Kinesin, dynein, ormyosin is engineered to contain a linking domain (e.g., a bindingdomain, e.g., such as a streptavidin binding domain) and incubated witha molecule or molecules that mediate linking a nucleic acid such as aDNA to the kinesin, dynein, or myosin. For example, the kinesin, dynein,or myosin comprisisg a streptavidin binding domain is incubated withstreptavidin and a biotinylated oligonucleotide that is complementary toa generic adaptor sequence used to make a DNA library (e.g., each DNA ofthe library comprises the adaptor sequence). The adaptor sequence issingle stranded and binds the complementary oligonucleotide attached tothe motor protein (e.g., myosin, kinesin, or dynein). TheDNA/oligonucleotide-motor protein complex is loaded onto the ZMWcontaining a microtubule or actin filament and the previously attachedpolymerase. The motor protein attaches to the microtubule or the actinfilament and transports the DNA library molecule to the bottom of theZMW where the polymerase is located. The polymerase binds the primedtemplate and sequencing begins.

Actin filaments and microtubules are dynamic structures comprisingsubunits that can be stabilized with chemical compounds. In someembodiments, actin filaments are stabilized with phalloidins, which bindactin filaments and prevent depolymerization. In some embodiments,microtubules are stabilized with paclitaxel, which has been shown toprovide microtubules that are stabilized for times of approximately aweek. After delivery, some embodiments provide that the actin filamentor microtubule structures are disrupted using compounds such ascytochalasin, leaving only the polymerase/nucleic acid complex in thewell of the ZMW. Or, the structure is left intact in some embodiments toanchor the polymerase/nucleic complex in the desired site.

Cytochalasins are fungal metabolites that have the ability to bind toactin filaments and block polymerization and the elongation of actin.Actin microfilaments have been widely studied using cytochalasins. Dueto their chemical nature, cytochalasins can help researchers understandthe importance of actin in various biological processes. The use ofcytochalasins has allowed researchers to understand actinpolymerization, cell motility, ruffling, cell division, contraction, andcell stiffness. The use of cytochalasins has been so important tounderstanding cytoskeletal movement and many other biological processes,researchers have created two synthetic cytochalasins. Paclitaxel is amitotic inhibitor that was isolated from the bark of the Pacific yewtree, Taxus brevifolia, from which its original tame “taxol” wasderived. When it was developed commercially, the generic name waschanged to paclitaxel and the commercial compound was sold under thetrademark TAXOL. In this formulation, paclitaxel is dissolved inCremophor EL and ethanol, as a delivery agent. A newer formulation, inwhich paclitaxel is bound to albumin, is sold under the trademarkABRAXANE. Paclitaxel stabilizes microtubules and as a result interfereswith the normal breakdown of microtubules during cell division. Togetherwith docetaxel, it forms the drug category of the taxanes. It was thesubject of a notable total synthesis. Phalloidin is one of a group oftoxins from the death cap (Amanita phalloides) known as phallotoxins.Phalloidin binds F-actin, preventing actin depolymerization.

The technology finds use in DNA sequencing, e.g., single moleculesequencing. Single molecule sequencing systems, e.g., as developed byPacific Biosciences are described in Voelkerding et al., 55 ClinicalChem: 641-58, 2009; MacLean et al., 7 Nature Rev. Microbiol.: 287-96;and in U.S. Pat. Nos. 7,170,050; 7,302,146; 7,313,308; and 7,476,503;all of which are herein incorporated by reference. This technologyutilizes reaction wells 50-100 nm in diameter and encompassing areaction volume of approximately 20 zeptoliters (10⁻²¹ liters).Sequencing reactions are performed using immobilized template, modifiedphi29 DNA polymerase, and high local concentrations of fluorescentlylabeled dNTPs. High local concentrations and continuous reactionconditions allow incorporation events to be captured in real time byfluor signal detection using laser excitation, an optical waveguide, anda CCD camera.

In certain embodiments, the technology finds use for the single moleculereal time (SMRT) DNA sequencing methods using zero-mode waveguides(ZMWs) developed by Pacific Biosciences or similar methods. With thistechnology, DNA sequencing is performed on SMRT chips, each containingthousands of zero-mode waveguides (ZMWs). A ZMW is a hole, tens ofnanometers in diameter, fabricated in a 100 nm metal film deposited on asilicon dioxide substrate. Each ZMW becomes a nanophotonic visualizationchamber providing a detection volume of just 20 zeptoliters (10⁻²¹ L).At this volume, the activity of a single molecule can be detectedamongst a background of thousands of labeled nucleotides. The ZMWprovides a window for watching DNA polymerase as it performs sequencingby synthesis. Within each chamber, a single DNA polymerase molecule isattached to the bottom surface such that it permanently resides withinthe detection volume. Phospholinked nucleotides, each type labeled witha different colored fluorophore, are then introduced into the reactionsolution at high concentrations which promote enzyme speed, accuracy,and processivity. Due to the small size of the ZMW, even at these high,biologically relevant concentrations, the detection volume is occupiedby nucleotides only a small fraction of the time. In addition, visits tothe detection volume are fast, lasting only a few microseconds, due tothe very small distance that diffusion has to carry the nucleotides. Theresult is a very low background.

While particular embodiments are described herein in reference toparticular DNA sequencing methods such as Single Molecule Real Time DNAsequencing as implemented by technologies developed by PacificBiosciences, the technology of delivering a molecule or macromolecule(e.g., a polymerase or DNA) to a site finds use in other sequencingtechnologies.

In some embodiments, the technology provided herein finds use in aSecond Generation (a.k.a. Next Generation or Next-Gen), Third Generation(a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencingtechnology including, but not limited to, pyrosequencing,sequencing-by-ligation, single molecule sequencing,sequence-by-synthesis (SBS), massive parallel clonal, massive parallelsingle molecule SBS, massive parallel single molecule real-time, massiveparallel single molecule real-time nanopore technology, etc. Morozovaand Marra provide a review of some such technologies in Genomics, 92:255 (2008), herein incorporated by reference in its entirety. Those ofordinary skill in the art will recognize that because RNA is less stablein the cell and more prone to nuclease attack experimentally RNA isusually reverse transcribed to DNA before sequencing.

A number of DNA sequencing techniques are known in the art, includingfluorescence-based sequencing methodologies (See, e.g., Birren et al.,Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; hereinincorporated by reference in its entirety). In some embodiments, thetechnology finds use in automated sequencing techniques understood inthat art. In some embodiments, the present technology finds use inparallel sequencing of partitioned amplicons (PCT Publication No:WO2006084132 to Kevin McKernan et al., herein incorporated by referencein its entirety). In some embodiments, the technology finds use in DNAsequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat.No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 toMacevicz et al., both of which are herein incorporated by reference intheir entireties). Additional examples of sequencing techniques in whichthe technology finds use include the Church polony technology (Mitra etal., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005Science 309, 1728-1732; U.S. Pat. Nos. 6,432,360, 6,485,944, 6,511,803;herein incorporated by reference in their entireties), the 454 picotiterpyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380;US 20050130173; herein incorporated by reference in their entireties),the Solexa single base addition technology (Bennett et al., 2005,Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; 6,833,246;herein incorporated by reference in their entireties), the Lynxmassively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5,695,934;5,714,330; herein incorporated by reference in their entireties), andthe Adessi PCR colony technology (Adessi et al. (2000). Nucleic AcidRes. 28, E87; WO 00018957; herein incorporated by reference in itsentirety).

Next-generation sequencing (NGS) methods share the common feature ofmassively parallel, high-throughput strategies, with the goal of lowercosts in comparison to older sequencing methods (see, e.g., Voelkerdinget al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; each herein incorporated by reference in theirentirety). NGS methods can be broadly divided into those that typicallyuse template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, technologies of OxfordNanopore Technologies Ltd., technologies of Life Technologies/IonTorrent, and the Supported Oligonucleotide Ligation and Detection(SOLiD) platform commercialized by Applied Biosystems. Non-amplificationapproaches, also known as single-molecule sequencing, are exemplified bythe HeliScope platform commercialized by Helicos BioSciences andemerging platforms commercialized by VisiGen and Pacific Biosciences.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. Nos.6,210,891; 6,258,568; each herein incorporated by reference in itsentirety), template DNA is fragmented, end-repaired, ligated toadaptors, and clonally amplified in-situ by capturing single templatemolecules with beads bearing oligonucleotides complementary to theadaptors. Each bead bearing a single template type is compartmentalizedinto a water-in-oil microvesicle, and the template is clonally amplifiedusing a technique referred to as emulsion PCR. The emulsion is disruptedafter amplification and beads are deposited into individual wells of apicotitre plate functioning as a flow cell during the sequencingreactions. Ordered, iterative introduction of each of the four dNTPreagents occurs in the flow cell in the presence of sequencing enzymesand luminescent reporter such as luciferase. In the event that anappropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10⁶ sequence readscan be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. Nos. 6,833,246; 7,115,400; 6,969,488; each herein incorporated byreference in its entirety), sequencing data are produced in the form ofshorter-length reads. In this method, single-stranded fragmented DNA isend-repaired to generate 5′-phosphorylated blunt ends, followed byKlenow-mediated addition of a single A base to the 3′ end of thefragments. A-addition facilitates addition of T-overhang adaptoroligonucleotides, which are subsequently used to capture thetemplate-adaptor molecules on the surface of a flow cell that is studdedwith oligonucleotide anchors. The anchor is used as a PCR primer, butbecause of the length of the template and its proximity to other nearbyanchor oligonucleotides, extension by PCR results in the “arching over”of the molecule to hybridize with an adjacent anchor oligonucleotide toform a bridge structure on the surface of the flow cell. These loops ofDNA are denatured and cleaved. Forward strands are then sequenced withreversible dye terminators. The sequence of incorporated nucleotides isdetermined by detection of post-incorporation fluorescence, with eachfluor and block removed prior to the next cycle of dNTP addition.Sequence read length ranges from 36 nucleotides to over 50 nucleotides,with overall output exceeding 1 billion nucleotide pairs per analyticalrun.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. Nos. 5,912,148; 6,130,073; each hereinincorporated by reference in their entirety) also involves fragmentationof the template, ligation to oligonucleotide adaptors, attachment tobeads, and clonal amplification by emulsion PCR. Following this, beadsbearing template are immobilized on a derivatized surface of a glassflow-cell, and a primer complementary to the adaptor oligonucleotide isannealed. However, rather than utilizing this primer for 3′ extension,it is instead used to provide a 5′ phosphate group for ligation tointerrogation probes containing two probe-specific bases followed by 6degenerate bases and one of four fluorescent labels. In the SOLiDsystem, interrogation probes have 16 possible combinations of the twobases at the 3′ end of each probe, and one of four fluors at the 5′ end.Fluor color, and thus identity of each probe, corresponds to specifiedcolor-space coding schemes. Multiple rounds (usually 7) of probeannealing, ligation, and fluor detection are followed by denaturation,and then a second round of sequencing using a primer that is offset byone base relative to the initial primer. In this manner, the templatesequence can be computationally re-constructed, and template bases areinterrogated twice, resulting in increased accuracy. Sequence readlength averages 35 nucleotides, and overall output exceeds 4 billionbases per sequencing run.

In certain embodiments, the technology finds use in nanopore sequencing(see, e.g., Astier et al., J. Am. Chem. Soc. 2006 Feb. 8;128(5):1705-10, herein incorporated by reference). The theory behindnanopore sequencing has to do with what occurs when a nanopore isimmersed in a conducting fluid and a potential (voltage) is appliedacross it. Under these conditions a slight electric current due toconduction of ions through the nanopore can be observed, and the amountof current is exceedingly sensitive to the size of the nanopore. As eachbase of a nucleic acid passes through the nanopore, this causes a changein the magnitude of the current through the nanopore that is distinctfor each of the four bases, thereby allowing the sequence of the DNAmolecule to be determined.

In certain embodiments, the technology finds use in HeliScope by HelicosBioSciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. Nos.7,169,560; 7,282,337; 7,482,120; 7,501,245; 6,818,395; 6,911,345;7,501,245; each herein incorporated by reference in their entirety).Template DNA is fragmented and polyadenylated at the 3′ end, with thefinal adenosine bearing a fluorescent label. Denatured polyadenylatedtemplate fragments are ligated to poly(dT) oligonucleotides on thesurface of a flow cell. Initial physical locations of captured templatemolecules are recorded by a CCD camera, and then label is cleaved andwashed away. Sequencing is achieved by addition of polymerase and serialaddition of fluorescently-labeled dNTP reagents. Incorporation eventsresult in fluor signal corresponding to the dNTP, and signal is capturedby a CCD camera before each round of dNTP addition. Sequence read lengthranges from 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub.Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073,and 20100137143, incorporated by reference in their entireties for allpurposes). A microwell contains a template DNA strand to be sequenced.Beneath the layer of microwells is a hypersensitive ISFET ion sensor.All layers are contained within a CMOS semiconductor chip, similar tothat used in the electronics industry. When a dNTP is incorporated intothe growing complementary strand a hydrogen ion is released, whichtriggers a hypersensitive ion sensor. If homopolymer repeats are presentin the template sequence, multiple dNTP molecules will be incorporatedin a single cycle. This leads to a corresponding number of releasedhydrogens and a proportionally higher electronic signal. This technologydiffers from other sequencing technologies in that no modifiednucleotides or optics are used. The per-base accuracy of the Ion Torrentsequencer is ˜99.6% for 50 base reads, with ˜100 Mb generated per run.The read-length is 100 base pairs. The accuracy for homopolymer repeatsof 5 repeats in length is ˜98%. The benefits of ion semiconductorsequencing are rapid sequencing speed and low upfront and operatingcosts.

The technology finds use in another nucleic acid sequencing approachdeveloped by Stratos Genomics, Inc. and involves the use of Xpandomers.This sequencing process typically includes providing a daughter strandproduced by a template-directed synthesis. The daughter strand generallyincludes a plurality of subunits coupled in a sequence corresponding toa contiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 20090035777, entitled “HighThroughput Nucleic Acid Sequencing by Expansion,” filed Jun. 19, 2008,which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

Other embodiments provide for the delivery of a molecule ormacromolecule to a site for an assay. Assays for which the technologyfinds use are, e.g., an ELISA or other immunoassay, array assays(nucleic acid or protein detection microarrays), etc.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EXAMPLES Example 1

Embodiments of the technology comprise a DNA polymerase engineered tocontain a kinesin binding domain as depicted in FIG. 1. The engineeredDNA polymerase is complexed with a DNA that is to be sequenced. The DNApolymerase/DNA is complexed with a kinesin and the DNApolymerase/DNA/kinesin complex is loaded into the zero mode waveguide.Upon the addition of ATP, the kinesin motor travels down the microtubuleto the bottom of the zero mode waveguide, in the desired site forsequencing. The DNA polymerase is anchored in the well either byremaining connected to the kinesin or by another interaction such as astreptavidin-biotin interaction used as an anchor.

Example 2

Embodiments of the technology comprise use of a chromokinesin (e.g., aKIN N chromokinesin) as depicted in FIG. 2. DNA libraries areconstructed to contain a chromokinesin binding sequence in an adapter.DNA is incubated with the chromokinesin to form a chromokinesin/DNAcomplex. The chromokinesin/DNA complex is loaded onto a zero modewaveguide, which has been preloaded with immobilized DNA polymerase atthe bottom of the well. Upon the addition of ATP, the chromokinesinmotor protein travels down the microtubule to the bottom of the zeromode waveguide. The DNA is delivered to the immobilized DNA polymerase.The microtubule and chromokinesin/DNA complex is disrupted, freeing theDNA to bind the polymerase.

Example 3

Embodiments of the technology comprise use of a kinesin engineered tocontain a streptavidin binding domain as depicted in FIG. 3. Kinesin isincubated with streptavidin and a biotinylated oligonucleotide that iscomplementary to the adaptor sequence of the DNA library. TheDNA/kinesin complex is formed via oligonucleotide/adaptor sequencebinding. The DNA/kinesin complex is loaded into the zero mode waveguide,which has been preloaded with immobilized DNA polymerase at the bottomof the well. Upon the addition of ATP, the kinesin moter protein travelsdown the microtubule to the bottom of the zero mode waveguide. The DNAis delivered to the immobilized DNA polymerase. Sequencing can begin onthe primed DNA template.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology, genomics, biochemistry, medical science, materialsscience, or related fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A method for delivering a macromolecule to an assay site,wherein the method comprises: 1) maintaining an end of a transport guideat the assay site; 2) providing a molecular motor adapted for bindingthe transport guide and moving along the transport guide; and 3) linkingthe macromolecule to the molecular motor.
 2. The method of claim 1,wherein the assay site is in a zero-mode waveguide or in a nanowell. 3.The method of claim 1, wherein the assay site comprises an anchor tomaintain the macromolecule at the assay site.
 4. The method of claim 1,wherein: a) the transport guide is a microtubule and the molecular motoris selected from the group consisting of a kinesin and a dynein; or b)the transport guide is an actin filament and the molecular motor is amyosin.
 5. The method of claim 1, wherein the macromolecule comprises aDNA polymerase and/or a DNA.
 6. The method of claim 1, furthercomprising providing a DNA polymerase at the assay site.
 7. The methodof claim 1, wherein maintaining the end of the transport guide at theassay site comprises attaching the end of the transport guide to theassay site.
 8. The method of claim 1, wherein the linking comprisesproviding a domain selected from the group consisting of a myosinbinding domain and a microtubule associated protein binding domain. 9.The method of claim 1, wherein the transport guide is stabilized with acomposition selected from the group consisting of a phalloidin and apaclitaxel.
 10. The method of claim 1, wherein the transport guide isdisassembled and/or destabilized by a cytochalasin.
 11. The method ofclaim 1, wherein the linking is a covalent interaction with themolecular motor.
 12. The method of claim 1, wherein the linking is anon-covalent interaction with the molecular motor.
 13. The method ofclaim 1, wherein the linking comprises a streptavidin and/or a biotin.